1. Field of the Invention
This invention relates generally to the systemic delivery of a therapeutic molecule via a liposome complex that is targeted to a pre-selected cell type. More specifically, the invention provides compositions and methods for cell-targeted gene transfer and gene therapy for human cancers whereby a therapeutic molecule is delivered to the targeted cancer cell via a ligand/liposome complex. Treatment of cell proliferative disease (e.g. cancer) results in substantial improvement of the efficacy of radiation and chemotherapeutic interventions.
2. Description of Related Art
The ideal therapeutic for cancer would be one that selectively targets a cellular pathway responsible for the tumor phenotype and would be nontoxic to normal cells. To date, the ideal therapeutic remains just thatxe2x80x94an ideal. While cancer treatments involving gene therapy and anti-sense molecules have substantial promise, there are many issues that need to be addressed before this promise can be realized. Perhaps foremost among the issues associated with macromolecular treatments for cancer and other diseases is the efficient delivery of the therapeutic molecule(s) to the site(s) in the body where they are needed.
A variety of nucleic acid delivery systems (xe2x80x9cvectorsxe2x80x9d) to treat cancer have been evaluated by others, including viruses and liposomes. The ideal vector for human cancer gene therapy would be one that could be systemically administered and then specifically and efficiently target tumor cells wherever they occur in the body. Viral vector-directed methods show high gene transfer efficiency but are deficient in several areas. The limitations of a viral approach are related to their lack of targeting and to the presence of residual viral elements that can be immunogenic, cytopathic, or recombinogenic.
A major deficiency of viral vectors is the lack of cancer cell specificity. Absent tumor targeting capability, viral vectors are limited in use to direct, local delivery that does not have the capability to reach metastatic diseasexe2x80x94the ultimate cause of death for the majority of cancer patients.
The high titers achievable and the cell tropism that makes viruses attractive as gene therapy and gene transfer delivery vectors present some of their greatest deficiencies. Although the preparation of novel viruses with new targets for infection has been described in the literature, these vectors are problematic due to the need for growing virus to high titer. Consequently, a substantial amount of attention has been directed to non-viral vectors for the delivery of molecular therapeutics, including use in gene transfer and gene therapy.
Progress has been made toward developing non-viral, pharmaceutical formulations of genes for in vivo human therapy, particularly cationic liposome-mediated gene transfer systems. Cationic liposomes are composed of positively charged lipid bilayers and can be complexed to negatively charged, naked DNA by simple mixing of lipids and DNA such that the resulting complex has a net positive charge. The complex is easily bound and taken up by cells, with a relatively high transfection efficiency. Features of cationic liposomes that make them versatile and attractive for DNA delivery include: simplicity of preparation, the ability to complex large amounts of DNA, versatility in use with any type and size of DNA or RNA, the ability to transfect many different types of cells (including non-dividing cells) and lack of immunogenicity or biohazardous activity. The liposome approach offers a number of advantages over viral methodologies for gene delivery. Most significantly, since liposomes are not infectious agents capable of self-replication, they pose no risk of evolving into new classes of infectious human pathogens. Further, cationic liposomes have been shown to be safe and somewhat efficient for in vivo gene delivery. Since liposomes are not infectious agents, they can be formulated by simple mixing. Further, cationic liposomes have been shown to be safe and somewhat efficient for in vivo gene delivery. Clinical trials are now underway using cationic liposomes for gene delivery, and liposomes for delivery of small molecule therapeutics (e.g., chemotherapeutic and antifungal agents) are already on the market.
One disadvantage of cationic liposomes is that they lack tumor specificity and have relatively low transfection efficiencies as compared to viral vectors. However, targeting cancer cells via liposomes can be achieved by modifying the liposomes so that they bear a ligand recognized by a cell surface receptor. Receptor-mediated endocytosis represents a highly efficient internalization pathway in eukaryotic cells. The presence of a ligand on a liposome facilitates the entry of DNA into cells through initial binding of ligand by its receptor on the cell surface followed by internalization of the bound complex. Once internalized, sufficient DNA can escape the endocytic pathway to be expressed in the cell nucleus.
There now exists a substantial knowledge base regarding the molecules that reside on the exterior surfaces of cancer cells. Surface molecules can be used to selectively target liposomes to tumor cells, because the molecules that are found upon the exterior of tumor cells differ from those on normal cells. For example, if a liposome has the protein transferrin (Tf) on its surface, it can target cancer cells that have high levels of the transferrin receptor.
A variety of ligands have been examined for their liposome-targeting ability, including folic acid (folate), a vitamin necessary for DNA synthesis, and transferrin. Both the folate receptor and transferrin receptor levels are found to be elevated in various types of cancer cells including ovarian, oral, breast, prostate and colon. The presence of such receptors can correlate with the aggressive or proliferative status of tumor cells. The folate receptor has also been shown to recycle during the internalization of folate in rapidly dividing cells such as cancer cells. Moreover, the transferrin and folate-conjugated macromolecules and liposomes have been shown to be taken up specifically by receptor-bearing tumor cells by receptor mediated endocytosis. Thus the folate and transferrin receptors are considered to be useful as prognostic tumor markers for cancer and as potential targets for drug delivery in the therapy of malignant cell growth.
Failure to respond to radiotherapy and chemotherapy represents an unmet medical need in the treatment of many types of cancer. Often, when cancer recurs, the tumors have acquired increased resistance to radiation or to chemotherapeutic agents. The incorporation into cancer therapies of a new component which results in sensitization to these therapies would have immense clinical relevance. One way in which such chemo/radio sensitization could be achieved is via targeted gene therapy.
An important role for p53 in the control of cellular proliferation by the regulation of cell cycle events and induction of programmed cell death (apoptosis) has been established. Since it appears that most anti-cancer agents work by inducing apoptosis, inhibition of, or changes in, this pathway may lead to failure of therapeutic regimens. A direct link, therefore, has been suggested between abnormalities in p53 and resistance to cytotoxic cancer treatments (both chemo- and radiotherapy). It has also been suggested that the loss of p53 function may contribute to the cross-resistance to anti-cancer agents observed in some tumor cells. Various groups have established a positive correlation between the presence of mutant p53 and chemoresistance in mouse fibrosarcomas and in primary tumor cultures from breast carcinomas, human gastric and esophageal carcinomas, as well as B-cell chronic lymphoblastic leukemia. In addition, chemosensitivity via apoptosis reportedly was restored by expression of wtp53 in non-small cell lung carcinoma mouse xenografts carrying mutant p53.
A role for the tumor suppressor gene p53 in many critical cellular pathways, particularly in the cellular response to DNA damage, has been established. These pathways not only include gene transcription, DNA repair, genomic stability, chromosomal segregation and senescence, but also regulation of cell cycle events and the modulation of programmed cell death (apoptosis). For its role in monitoring DNA damage, p53 has been christened xe2x80x9cguardian of the genome.xe2x80x9d Cancer cells are characterized by genetic instability, and mutations in p53 have been found to occur with extremely high frequency in almost all types of human cancers. Indeed, quantitative or qualitative alterations in the p53 gene are suggested to play a role in over half of all human malignancies. The presence of p53 mutations in the most common types of human tumors has been found to be associated with poor clinical prognosis. Moreover, mutant (mt) p53 is rarely found in some of the more curable forms of cancer e.g., Wilms""s tumor, retinoblastoma, testicular cancer, neuroblastoma and acute lymphoblastic leukemia.
Numerous studies have reported that the expression of wt p53 has suppressed, both in vitro and in mouse xenograft models, the growth of various malignancies, e.g., prostate, head and neck, colon, cervical and lung tumor cells. It has also been reported that a p53-liposome complex partially inhibited the growth of human glioblastoma and human breast cancer xenografts in mice. In addition, Seung et al. used liposome-mediated intratumoral introduction of a radiation-inducible construct expressing TNF-xcex1 to inhibit growth of a murine fibrosarcoma xenograft after exposure to ionizing radiation. However, p53 expression alone, while being able to inhibit tumor growth partially, has not been able to eliminate established tumors in the long-term.
The normal development of mice lacking wtp53 and the observations of a post-irradiation G1 block in p53-expressing cells suggests that wt p53 functions in the regulation of the cell after DNA damage or stress rather than during proliferation and development. Since it appears that many conventional anti-cancer therapies (chemotherapeutics and radiation) induce DNA damage and appear to work by inducing apoptosis, alterations in the p53 pathway could conceivably lead to failure of therapeutic regimens.
Lack of wt p53 function has also been associated with an increase in radiation resistance. The presence of mt p53 and the consequent absence of a G1 block have also been found to correlate with increased radiation resistance in some human tumors and cell lines. These include human tumor cell lines representative of head and neck, lymphoma, bladder, breast, thyroid, ovary and brain cancer.
Based on these considerations, gene therapy to restore wtp53 function in tumor cells should re-establish the p53-dependent cell cycle checkpoints and the apoptotic pathway thus leading to the reversal of the chemo-/radio-resistant phenotypes. Consistent with this model, chemosensitivity, along with apoptosis, was restored by expression of wtp53 in non-small cell lung carcinoma mouse xenografts carrying mtp53. Chemosensitivity of xenografts involving the p53-null lung tumor cell line H1299 and T98G glioblastoma cells and sensitivity of WiDr colon cancer xenografts to cisplatin has been demonstrated. Increased cell killing by doxorubicin or mitomycin C was also shown in SK-Br-3 breast tumor cells by adenoviral transduction of wtp53. However, some conflicting reports indicate that the relationship between p53 expression and chemoresistance may have a tissue or cell type-specific component. The transfection of wtp53 by an adenoviral vector has also been shown to sensitize ovarian and colo-rectal tumor cells to radiation It has also been reported that adenoviral-mediated wtp53 delivery did restore functional apoptosis in a radiation-resistant squamous cell carcinoma of the head and neck (SCCHN) tumor line resulting in radiosensitization of these cells in vitro. More significantly, the combination of intratumorally injected adeno-wtp53 and radiation led to complete and long-term tumor regression of established SCCHN xenograft tumors.
The current invention departs from the conventional use of viral vectors for the delivery of therapeutic molecules for gene therapy, for example as disclosed by Roth et al. (U.S. Pat. No. 5,747,469). These currently used vehicles only have the limited capability of local delivery. Their suitability for intratumoral delivery has been shown not only to be inadequate in reaching all of the cells within the primary tumor mass, but also incapable of reaching sites of metastatic disease.
In one aspect, the invention provides cell-targeting ligand/liposome/therapeutic molecule complexes for the in vitro or in vivo delivery of therapeutic molecules to targeted cell types. The complexes are useful as delivery vehicles (vectors) for delivering a therapeutic molecule to the target cells. The complexes are useful as vectors for carrying out gene transfer and gene therapy when the therapeutic molecule is, for example, a nucleic acid encoding a therapeutic protein. Specific embodiments relate to folate and transferrin-targeted cationic liposomes for the delivery of a therapeutic molecule to animal (including human) cancer cells that contain folate or transferrin receptors.
In another aspect, the invention provides pharmaceutical compositions comprising a cell-targeting ligand/liposome/therapeutic molecule complex in a pharmaceutically compatible vehicle or carrier. The compositions are formulated for, preferably, intravenous administration to a human patient to be benefitted by the effective delivery of the therapeutic molecule. The complexes are appropriately sized so that they are distributed throughout the body following i.v. administration.
In another aspect, the invention relates to therapeutic methods comprising the administration to a warm-blooded animal (including humans) in need thereof, of a therapeutically effective amount of a pharmaceutical composition comprising a ligand/liposome/therapeutic molecule complex in a pharmaceutically acceptable vehicle. As set forth in detail herein, human cancer treatment via the systemic (e.g. i.v.) administration of a complex comprising a ligand-targeted liposome complex containing a nucleic acid encoding wt p53 is an important embodiment of this aspect of the invention.
Human gene therapy via the systemic administration of pharmaceutical compositions containing targeted liposome/nucleic acid complexes, wherein the nucleic acid comprises a therapeutic gene under the control of an appropriate regulatory sequence, form important examples of the invention. Gene therapy for many forms of human cancers is accomplished by the systemic delivery of folate or transferrin-targeted cationic liposomes containing a nucleic acid encoding wt p53. The data presented herein demonstrates the superior ability of such complexes to specifically target and sensitize tumor cells (due to expression of the wt p53 gene), both primary and metastatic tumors, to radiation and/or chemotherapy both in vitro and in vivo.
Yet another aspect of the invention relates to improvements to the preparation of liposomes, especially ligand-targeted cationic liposomes, whereby liposomes of relatively small, consistent diameters are provided. The consistent, small-diameter liposomes, following intravenous administration, exhibit the ability to circulate in the bloodstream and target both primary tumors and metastases.
The present invention addresses the need to deliver therapeutic molecules systemically with a high degree of target cell specificity and high efficiency. When systemically administered, the complexes of the present invention are capable of reaching, and specifically targeting, metastatic as well as primary disease, when the target cells are human cancer cells. As a result of delivery of the normal, wild-type version of the tumor suppressor gene p53 by means of this system, the inventors demonstrated that the tumors are sensitized to radiation therapy and/or chemotherapy. The high transfection efficiency of this system results in such a high degree of sensitization that not only is there growth inhibition of the cancer but pre-existing tumors and metastases are completely eliminated for an extended period of time. In some instances this period of time is such that the disease may be considered to be cured.
The exceptional efficacy of this system is due in part to the ligand-targeting of the liposome-therapeutic molecule complex. Moreover, the specific cationic and neutral lipids that comprise the liposome complex, as well as the ratio of each, have been varied and optimized so that the efficiency of uptake of the therapeutic molecule would be ideal for the specific target cell type. The ratio of liposome to therapeutic molecule was also optimized for target cell type. This optimization of the liposome-therapeutic molecule complex, in combination with the addition of a targeting ligand, yields substantially improved efficacy when administered in conjunction with radiation or chemotherapies. Those skilled in the art will be able to optimize the complexes for delivering a variety of therapeutic molecules to a variety of cell types.
An important feature of the invention resides in the ability to deliver the therapeutic molecule to the target cell through intravenous, systemic administration. The ability to efficiently target and transfect specific cells following intravenous administration is accomplished by the disclosed combination of selecting an appropriate targeting ligand and optimizing the ratio of cationic to neutral lipid in the liposome. In the case where tumor cells are the target cells, systemic delivery of the ligand-liposome-therapeutic molecule complex allows the efficient and specific delivery of the therapeutic molecule to metastases as well as primary tumor.
The invention is not limited to the use of any specific targeting ligand. The ligand can be any ligand for which a receptor is differentially expressed on the target cell. The presently preferred ligands are folic acid (esterified to a lipid of the liposome) and transferrin, and each of these ligands possesses advantageous properties.
Liposome complexes are capable of penetrating only approximately twenty layers of cells surrounding the blood vessels in a tumor. It has been postulated that wtp53 gene therapy controls cell growth partially through a xe2x80x9cbystanderxe2x80x9d effect, which may be related to the induction of apoptosis by wtp53. This xe2x80x9cbystander effectxe2x80x9d may account for the effectiveness of the in vivo studies reported herein and may be a contributory factor to the effectiveness of the combination therapy. However, relatively little is known at this time concerning the mechanism and pathway involved in this process for p53. It has been speculated that some as yet unknown apoptotic signal may be contained within the vesicles, which result from apoptosis, and which neighboring cells ultimately phagocytize. Alternatively, this apoptotic signal may be transferred through gap junctions, as is believed to be the case for phosphorylated gancyclovir with the HSV-TK gene. Induction of anti-angiogenic factors may also contribute to the bystander effect.
It has recently been reported that a non-targeted p53-liposome complex partially inhibited the growth of human glioblastoma xenografts in vivo. In addition, Seung et al. (Cancer Res. 55, 5561-5565 (1995) used commercial non-targeted liposome (Lipofectin) mediated intratumoral introduction of a radiation inducible construct containing TNF-xcex1 to partially inhibit xenograft growth of a murine fibrosarcoma after exposure to 40 Gy ionizing radiation. Xu et al. (Human Gene Therapy 8, 177-175 (1997)) showed that introduction of 16 xcexcg of p53 DNA in a non-targeted liposome complex was able to partially inhibit the growth of breast cancer xenograft mouse tumors. However, the ligand-directed liposome-p53 complexes of the present invention provide the capacity for target cell specificity, and high transfection efficiency, coupled with systemic administration. The studies reported here are the first to employ such a delivery system in combination with conventional radiation and chemotherapeutic treatment for tumors. While p53 gene therapy alone may not be sufficient to completely eliminate tumors long term, the presently-described combination of liposome-mediated p53 gene therapy and conventional (radiation and/or chemotherapy) therapy was able to achieve not only growth inhibition, but tumor regression, demonstrating a synergistic effect.
The in vivo studies described herein demonstrate that the combination of systemic LipF-p53 or LipT-p53 gene therapy and conventional radiotherapy and/or chemotherapy was markedly more effective than either treatment alone. In the clinical setting, radiation doses of 65 to 75 Gy for gross tumor and 45 to 50 Gy for microscopic disease are commonly employed in the treatment of head and neck cancer. Given the known, adverse side effects associated with high doses of radiation or chemotherapy, sensitization of tumors so as to permit a lowered effective dose of the conventional treatment would be of immense clinical benefit. Furthermore, in the case of radiation, systemic restoration of wtp53 function, resulting in a decrease in the radiation treatment dose found to be effective, would permit further therapeutic intervention for tumors which did reoccur.
In reports using xenograft tumors derived from SCCHN cell lines containing either wtp53 or mtp53 it was noted that introduction of wtp53, via intratumoral administration of an adenoviral vector, was able to inhibit the development of, and induce apoptosis in, these xenograft tumors independent of their endogenous p53 status. Similarly, liposome-mediated introduction of wtp53 into both glioblastoma (RT-2) and breast cancer (MCF-7) xenografts, which have endogenous wtp53, was able to partially inhibit the growth of these tumors. These studies indicate the broad potential of wtp53 gene therapy, irrespective of p53 gene status.
The research underlying the present invention demonstrates that the ligand-cationic liposome-therapeutic molecule complex system can deliver the p53 gene in vivo selectively to tumors of various types, sensitizing them to radiation and chemotherapy. Consequently, systemic wtp53 gene therapy, mediated by the tumor-targeting, relatively safe and efficient ligand-targeted cationic liposome system, in combination with conventional radiotherapy or chemotherapy, may provide a more effective treatment modality not only for primary tumors, but also for those cancers which fail initial therapy.
It also has been demonstrated that the targeted liposome delivery system is capable of delivering small DNA molecules (e.g. antisense oligonucleotides), as well as agents as large as intact viral particles. Delivery of these small (antisense) DNA molecules was also able to sensitize tumor cells to chemotherapeutic agents. Thus, the targeted liposomes of the present invention are widely applicable to the systemic delivery of therapeutic agents.
The invention also relates to methods for preparing ligand-liposome-therapeutic agent complexes. The method by which the complex is formed between the transferrin-liposome and viral particle provides a large number of transferrin molecules upon the surface of the complex and thereby increases the stability of the complex as it travels through the blood stream. Moreover, when the therapeutic molecule is a viral particle, the transferrin liposome may also serve to decrease the immunogenicity of the virus by blocking viral antigens.
Using the present invention, the inventors have demonstrated a remarkable effect not only in controlling cell growth, in particular tumor cell growth, but also in effecting tumor remission long-term. Tumor cell formation and growth, also known as transformation, describes the formation and proliferation of cells that have lost their ability to control cell division, that is, they are cancerous. A number of different types of transformed cells can serve as targets for the methods and compositions of the present invention, such as: carcinomas, sarcomas, melanomas, and a wide variety of solid tumors and the like. Although any tissue having malignant cell growth may be a target, head and neck, breast, prostate, pancreatic, glioblastoma, cervical, lung, liposarcoma, rhabdomyosarcoma, choriocarcinoma, melanoma, retinoblastoma, ovarian, gastric and colorectal cancers are preferred targets.
It is further contemplated that the invention can also be used to target non-tumor cells for delivery of a therapeutic molecule. While any normal cell can be a target, the preferred normal targets are dendritic cells, endothelial cells of the blood vessels, lung cells, breast cells, bone marrow cells, and liver cells.
It is disclosed herein that, when delivered systemically, the ligand-targeted, optimized cationic liposomal-therapeutic molecule complex was able to specifically target and markedly sensitize tumor cells to radiation and/or chemotherapy resulting in substantial growth inhibition and tumor regression. The ligand-targeted, optimized cationic liposomal-therapeutic molecule complex may be delivered via other routes of administration such as intratumoral, aerosol, percutaneous, endoscopic, topical, intralesional or subcutaneous administration.
The invention provides, in certain embodiments, methods and compositions for the highly target cell-specific and efficient delivery, via systemic administration, of a ligand-targeted, liposomal-therapeutic molecule complex. Examples of therapeutic molecules include a gene, high molecular weight DNA, plasmid DNA, an antisense oligonucleotide, peptides, ribozymes, peptide nucleic acids, a chemical agent such as a chemotherapeutic molecule, or any large molecule including, but not limited to, DNA, RNA, viral particles, growth factors cytokines, immunomodulating agents and other proteins, including proteins which when expressed present an antigen which stimulates or suppresses the immune system.
Recently, efficient methods for long term expression of gene therapy vectors have been described (Cooper, et al, 1997; Westphal et al., 1998; Calos, 1996 and 1998). These vectors can be useful for extending and/or increasing the expression levels of the disclosed liposomal delivery system. Several autonomous and episomal vector systems are disclosed in U.S. Pat. Nos. 5,707,830 (Calos, M. P., 13 Jan. 1998); 5,674,703 (Woo, S., et al., 7 Oct. 1997) and 5,624,820 (Cooper, M. J., 29 Apr. 1997) each of which is incorporated by reference herein. Calos relates to Epstein Barr virus-based episomal expression vectors useful in autonomous replication in mammalian cells. Woo et al. relates to papilloma virus-based episomal expression vectors for replication in animal cells. Cooper et al. relates to vectors containing at least one papovavirus origin of replication and a mutant form of papovavirus large T antigen for long term episomal expression in human gene therapy.
When the therapeutic molecule is the p53 gene or an antisense oligonucleotide, delivery via the complex of the invention results in the sensitization of a cell or cells, such as a malignant cell or cells, to either radiation or a chemotherapeutic agent such that the cells are killed via the combination therapy. Malignant cells are defined as cells that have lost the ability to control the cell division cycle as leads to a transformed or cancerous phenotype. In addition to malignant cells, cells that may be killed using the invention include e.g., undesirable but benign cells, such as benign prostatic hyperplasia cells, over-active thyroid cells, lipoma cells, as well as cells relating to autoimmune diseases such as B cells that produce antibodies involved in arthritis, lupus, myasthenia gravis, squamous metaplasia, dysplasia and the like.
The ligand-liposome-therapeutic molecule complex can be formulated under sterile conditions within a reasonable time prior to administration. If the therapeutic molecule is one which provides enhanced susceptibility to another therapy (such as enhanced susceptibility of cancer cells to chemotherapy or radiation therapy), such other therapy may be administered before or subsequent to the administration of the complex, for example within 12 hr to 7 days. A combination of therapies, such as both chemotherapy and radiation therapy, may be employed in addition to the administration of the complex.
The terms xe2x80x9ccontactedxe2x80x9d or xe2x80x9cexposedxe2x80x9d when applied to a cell are used herein to describe the process by which a therapeutic molecule is delivered to a cell, or is placed in direct juxtaposition with the target cell, so that it can effectively interact with the cell to bring about a desired benefit to the cell or the host animal.
Wherein the complexes of the invention are used as an element of a combination therapy for, for example, human cancer treatment, they may be used in combination with a wide variety of therapies employed in the treatment of human or animal cancer. Such therapies include the administration of chemotherapeutic agents and radiation therapies such as gamma-irradiation, X-rays, UV irradiation, microwaves, electronic emissions and the like. Chemotherapeutic agents such as doxorubicin, 5-fluorouracil (5FU), cisplatin (CDDP), docetaxel, gemcitabine, pacletaxel, vinblastine, etoposide (VP-16), camptothecia, actinomycin-D, mitoxantrone and mitomycin C can be employed in combination therapies according to the present invention.
A variety of different types of potentially therapeutic molecules can be complexed with the cell-targeted ligand/liposome complexes of the invention. These include, but are not limited to, high molecular weight DNA molecules (genes), plasmid DNA molecules, small oligonucleotides, RNA, ribozymes, peptides, immunomodulating agents, peptide nucleic acids, viral particles, chemical agents such as per se known chemotherapeutic agents and drugs, growth factors, cytokines and other proteins including those which, when expressed, present an antigen which stimulates or suppresses the immune system. Therefore, in addition to gene therapy, the present invention can be used for immunotherapy or for the targeted delivery of drugs.
Diagnostic agents also can be delivered to targeted cells via the disclosed complexes. Agents which can be detected in vivo after administration to a multi-cellular organism can be used. Exemplary diagnostic agents include electron dense materials, magnetic resonance imaging agents and radiopharmaceuticals. Radionuclides useful for imaging include radioisotopes of copper, gallium, indium, rhenium, and technetium, including isotopes 64Cu, 67Cu, 111In, 99mTc, 67Ga or 68Ga. Imaging agents disclosed by Low et al. (U.S. Pat. No. 5,688,488) are useful in the present invention, and that patent is incorporated by reference herein.
The ligand-liposome composition of the invention, which will be complexed with the therapeutic molecule, can be comprised of a ligand, a cationic lipid and a neutral or helper lipid, where the ratio of cationic lipid to neutral lipid is about 1:(0.5-3), preferably 1:(1-2) (molar ratio). The ligand can be bound, e.g. via chemical coupling, to the neutral lipid and mixed with cationic lipid and neutral lipid at a molar ratio of about (0.1-20):100, preferably (1-10):100, and more preferably (2.5-5):100 (ligand-lipid:total lipids), respectively. The ligand-liposome will be mixed with DNA or other therapeutic molecules to form a complex. The DNA to lipid ratios will be in a range of about 1:(0.1-50), preferably about 1:(1-24), and more preferably about 1:(6-16) xcexcg/nmol. For antisense oligonucleotides, the complex will be formed by mixing the liposome with oligonucleotides at a molar ratio of about (5-30):1 lipid:oligonucleotide, preferably about (10-25):1, and most preferably about 10:1.
Alternatively, as in the case of transferrin, the ligand can simply be mixed with the cationic and neutral lipids. In this instance, the cationic liposomes will be prepared at a molar ratio of cationic lipid to neutral lipid of about 1:(0.5-3), preferably 1:(1-2). Transferrin will be mixed with the cationic liposomes and then DNA or other therapeutic molecules. The DNA/Lipid/Tf ratios will be in the range of about 1:(0.1-50):(0.1-100) xcexcg/nmol/xcexcg, preferably about 1:(5-24):(6-36), and more preferably about 1:(6-12):(8-15), respectively.
Another unique feature of the complexes according to the invention is their evenly distributed relatively small size (mean diameter less than about 100 nm, preferably less than about 75 nm, and more preferably about 35-75 nm (50 nm average) diameter). To reach the target tumor, the complexes must be resistant to degratory agents encountered in vivo, and also must be capable of passing through the blood vessel (capillary) walls and into the target tissue. The complexes of the present invention exhibit high resistance to degradation by elements present in serum. The permeable size of the capillaries in tumors is usually 50-75 nm, and the complexes which are less than about 75 nm diameter can pass easily through the capillary wall to reach the target. Based upon transmission electron microscopy, it appears that a unique onion-like layered structure of the LipF-DNA and LipT-DNA complex plays an important role in the small size and, consequently, high transfection efficiency of the complex of the invention observed in vitro and, in particular, in vivo.
The ligand can be any molecule that will bind to the surface of the target cell, but preferentially to a receptor that is differentially expressed on the target cell. Two particularly preferred ligands are folate and transferrin. The cationic lipid can be any suitable cationic lipid, but dioleoyltrimethylammonium-propane (DOTAP) and DDAB are preferred. The neutral lipid can be any neutral lipid, and preferred neutral lipids are dioleoylphosphatidylethanolamine (DOPE) and cholesterol.
A number of in vitro parameters may be used to determine the targeting and delivery efficiency of the composition so that particular complexes can be optimized to deliver a desired therapeutic molecule to the selected target cell type. These parameters include, for example, the expression of marker genes such as the xcex2-galactosidase or luciferase genes, immunohistochemical staining of target cells for the delivered protein, Western blot analysis of the expression of the protein product of the delivered gene, down-modulation of the target gene due to a delivered anti-sense or other inhibitory oligonucleotide, as well as increased sensitization of the target cells to radiation and/or chemotherapeutic agents.
In a preferred embodiment, it is contemplated that the p53 expression region will be positioned under the control of a strong constitutive promoter such as an RSV or a CMV promoter. Currently, a particularly preferred promoter is the cytomegalovirus (CMV) promoter.
The methods and compositions of the present invention are suitable for targeting a specific cell or cells in vitro or in vivo. When the target cells are located within a warm-blooded animal, e.g. head and neck, breast, prostate, pancreatic or glioblastoma cells, the ligand-liposome-therapeutic molecule complex will be administered to the animal in a pharmacologically acceptable form. A xe2x80x9cpharmacologically acceptable formxe2x80x9d, as used herein refers to both the formulation of the ligand-liposome-therapeutic molecule complex that may be administered to an animal, and also the form of contacting an animal with radiation, i.e. the manner in which an area of the animals body is irradiated, e.g. with gamma-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions and the like. The use of DNA damaging radiation and waves is known to those skilled in the art of radiation therapy.
The present invention also provides improved methods for treating cancer, both primary and metastatic, that, generally, comprise administering to an animal or human patient in need thereof a therapeutically effective combination of a ligand-liposome-therapeutic molecule (e.g. p53 gene) complex, and a therapy such as radiation or chemotherapy.
The complex will generally be administered to the animal, usually systemically, in the form of a pharmaceutically acceptable composition. In the preferred embodiment, the composition would be delivered systemically through an intravenous route. However, other routes of administration such as aerosol, intratumoral, intralesional, percutaneous, endoscopic, topical or subcutaneous may be employed.
The high degree of tumor cell specificity and tumor targeting ability of the invention was demonstrated by the expression of a reporter gene after systemic delivery by the folate/transferrin-liposome-xcex2-Gal gene complex. xcex2-galactosidase expression was evident in up to 70% of the xenografts of various human tumor cells, including JSQ-3, DU145 and MDA-MB-435, while normal tissues and organs, including the highly proliferative gut and bone marrow, showed no evidence of transfection. The highly efficient tumor targeting ability of the invention was also evident in these experiments where metastases, and even micro-metastases as small as a few cells, were found to have been specifically transfected after systemic delivery of the complex.
The surprising success of the present invention is evidenced by the finding that systemic delivery of either folate-liposome-wtp53 gene or transferrin-liposome-wtp53 gene, in combination with either radiation or chemotherapy, yielded profound results in studies using a nude mouse model. The high efficiency of this system results in such a high degree of sensitization of JSQ-3 and DU145 human xenograft tumors to radiation that not only is there growth inhibition of the cancer but, in some experiments, the pre-existing tumors and metastases were completely eliminated for an extended period of time. In some instances this period of time (more than one year disease-free) is such that the disease may be considered to be cured. Human breast cancer MDA-MB-435 and human pancreatic cancer PANC I nude mouse xenograft tumors were also shown to be highly sensitized by the systemic administration of either folate-liposome-wtp53 or transferrin-liposome-wtp53 to chemotherapeutic agents including doxorubicin, cisplatin, docetaxel or gemcitabine.
As used herein, the term xe2x80x9ctransfectionxe2x80x9d is used to describe the targeted delivery of a therapeutic molecule to eukaryotic cells using the ligand-liposome complex of the invention and entry of the therapeutic molecule into the cell by various methods, such as receptor mediated endocytosis. The target cell may be preferentially selected by the ligand of the complex such that the ligand will bind to a receptor that is differentially expressed on the surface of the target cell.
Preferred pharmaceutical compositions of the invention are those that include, within a pharmacologically acceptable solution or buffer, a complex consisting of a ligand, a cationic-neutral liposome and a therapeutic molecule.
Still further embodiments of the present invention are kits for use in the systemic delivery of a therapeutic molecule by the ligand-liposome complex, as may be formulated into therapeutic compositions for systemic administration. The kits of the invention will generally comprise, in separate, suitable containers, a pharmaceutical formulation of the ligand, of the liposome and of the therapeutic molecule. In the preferred embodiment the ligand would be either folate or transferrin, the liposome would consist of a cationic and a neutral lipid and the therapeutic molecule would be either a construct carrying wtp53 under control of the CMV promoter, or an antisense oligonucleotide. The three components can be mixed under sterile conditions and administered to the patient within a reasonable time frame, generally from 30 min to 24 hours, after preparation.
The components of the kit are preferably provided as. solutions or as dried powders. Components provided in solution form preferably are formulated in sterile water-for-injection, along with appropriate buffer(s), osmolarity control agents, antibiotics, etc. Components provided as dry powders can be reconstituted by the addition of a suitable solvent such as sterile water-for-injection.
The present invention uses systemic administration of a ligand/cationic liposomal delivery complex for tumor-targeted delivery of a therapeutic molecule via receptor-mediated endocytosis. In one of the preferred embodiments, the ligand-targeted liposomes are used to deliver a therapeutic molecule comprising a gene encoding wild-type (wt) p53. The therapeutic gene is targeted and effectively delivered to tumor cells, resulting in the restoration of the normal p53 gene function that many tumors lack. This restoration has a profound effect on the ability to treat the tumors. In another preferred embodiment, the therapeutic molecules being delivered are antisense oligonulceotides directed against genes in the cell growth pathway. Down-modulation of these genes results in sensitization of the tumor cells and xenografts to radiation and chemotherapeutic agents. In yet another embodiment, the xe2x80x9ctherapeutic moleculexe2x80x9d is an intact viral vector (e.g. an adenoviral or retroviral particle containing a therapeutic nucleic acid) which is delivered to the targeted cell via the ligand/liposome complex.
In another aspect, the invention provides compositions and methods for accomplishing gene therapy to restore wtp53 function in tumor cells, leading to the reversal of chemo-/radio-resistant phenotypes and consequently improving the ability to treat the tumor via chemo- and/or radiation therapy.
The present invention provides a new and improved method for accomplishing cancer gene therapy by providing a systemic delivery system (xe2x80x9ccomplexxe2x80x9d) that specifically targets tumor cells, including metastases, and results in a more effective cancer treatment modality. This method uses a ligand-directed cationic liposome system to deliver a therapeutic molecule to the tumor cells. In one of the preferred embodiments, this therapeutic molecule is wtp53. The inclusion of a cell-targeting ligand (e.g. the folate or transferrin ligand) in the liposome-DNA complex takes advantage of the tumor-targeting facet and receptor-mediated endocytosis associated with the ligand to introduce wtp53 efficiently and specifically to the tumor cells in vivo as well as in vitro. The consequence of this restoration of wtp53 function is an increase in sensitization to conventional radiation and chemo-therapies, thereby increasing their efficacy and/or reducing the total dose thereof.
The exemplified liposome compositions are based upon the cationic lipid DOTAP and fusogenic neutral lipid DOPE conjugated (e.g. esterified) to either folic acid (to provide a folate ligand thereon) or simply mixed with iron-saturated transferrin. The ratio of lipids themselves, as well as the lipid:DNA ratio, will be optimized for in vivo delivery, as well as for different tumor cell types, e.g. adenocarcinoma vs. squamous cell carcinoma. In vitro studies demonstrated that the addition of the ligand substantially increased the transfection efficiency for tumor cells when compared to the liposome alone, even in the presence of high levels of serum. Transfection of wtp53 by this method resulted in substantial radiosensitization of a previously radiation resistant SCCHN cell line in vitro.
The in vivo tumor targeting capability of this system was assessed using the xcex2-galactosidase reporter gene in three different types of cancerxe2x80x94SCCHN, breast cancer and prostate cancer. These studies demonstrated that after intravenous administration of the complexes, only the tumors were transfected, with an efficiency between 50 and 70%, while normal organs and tissues, including the highly proliferative bone marrow and intestinal crypt cells, showed no signs of reporter gene expression. Some ligand-liposome-DNA complex was evident in macrophages. Very significantly, even micro-metastases in the lung, spleen and lymph nodes showed evidence of highly efficient and specific transfection.
When the systemically delivered ligand-liposome wtp53 complex was administered to mice bearing radiation resistant human SCCHN xenografts, and followed with radiation therapy, the tumors completely regressed. Histological examination of the area of the former tumor showed only normal and scar tissue remaining, with no evidence of live tumor cells. This was in contrast to the tumors from animals treated only with the ligand-liposome-p53 complex or only with radiation. In these animals some cell death was evident. However, nests of live tumor cells remained, resulting in the regrowth of the tumors in these animals. Strikingly, no recurrence of the tumors was evident in the animals receiving the combination therapy, even one year after the end of treatment. Similar results were observed in mice bearing human prostate tumor xenografts with radiation and chemotherapeutic agents, as well as with human breast cancer and pancreatic cancer xenografts with chemotherapeutic agents. Consequently, this system is viewed as providing a more effective form of cancer therapy.
Therefore, the present invention represents a significant improvement upon current experimental cancer therapies, such as local injection of adenoviral vectors carrying a therapeutic molecule such as p53, which are frequently incapable of administering a therapeutic molecule to the entire tumor tissue (primary tumor mass). Local delivery also lacks the capability of reaching distant metastases. The specific targeting ability provided by the present invention is also advantageous since it reduces side effects that can be associated with wide spread non-specific uptake of the therapeutic molecule.
The uptake of the ligand-liposome-therapeutic molecule complex by the target cells will, when administered in conjunction with adjuvant therapies, and when the target cells are cancer cells, not only decrease the rate of proliferation of these cells but actually result in increased tumor cell death and long-term tumor regression. The delivery system of the invention strongly portends a prolongation of patient survival.
Even though the invention has been described with a certain degree of particularity, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the present disclosure. Accordingly, it is intended that all such alternatives, modifications, and variations which fall within the spirit and the scope of the invention be embraced by the defined claims.